Guava Nexin Annexin V Assay

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	Cells were seeded in 6-well tissue culture plates the night before siRNA transfections. At 24 h post-siRNA addition, the cells were treated with nutlin-3b or 3a for additional 24–96 h. Culture media were collected and combined with the trypsinized-attached cells. Cell pellets were collected by centrifugation at 2000 r.p.m. for 10 min at 4 °C. The percentage of apoptotic cells was determined by Annexin V/7-AAD staining using the Guava Nexin kit and the Guava personal cell analyzer (Guava Technologies, Hayward, CA, USA), and calculated as the total fraction Annexin V-positive cells as described (Thompson et al., 2004).

Protocol tips

Cells were seeded in 6-well tissue culture plates the night before siRNA transfections. At 24 h post-siRNA addition, the cells were treated with nutlin-3b or 3a for additional 24–96 h. Culture media were collected and combined with the trypsinized-attached cells. Cell pellets were collected by centrifugation at 2000 r.p.m. for 10 min at 4 °C. The percentage of apoptotic cells was determined by Annexin V/7-AAD staining using the Guava Nexin kit and the Guava personal cell analyzer (Guava Technologies, Hayward, CA, USA), and calculated as the total fraction Annexin V-positive cells as described (Thompson et al., 2004).

Publication protocol

Cells were seeded in 6-well tissue culture plates the night before siRNA transfections. At 24 h post-siRNA addition, the cells were treated with nutlin-3b or 3a for additional 24–96 h. Culture media were collected and combined with the trypsinized-attached cells. Cell pellets were collected by centrifugation at 2000 r.p.m. for 10 min at 4 °C. The percentage of apoptotic cells was determined by Annexin V/7-AAD staining using the Guava Nexin kit and the Guava personal cell analyzer (Guava Technologies, Hayward, CA, USA), and calculated as the total fraction Annexin V-positive cells as described (Thompson et al., 2004).

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